This invention relates to a method for the isolation of unreduced oligosaccharides from glycoproteins and glycohormones having an N-linked oligosaccharide structure.
The isolation of oligosaccharides from glycoproteins and glycohormones is of great interest for a number of reasons. First, it is of primary importance in the structural analysis of the carbohydrate moieties of the parent glycoproteins and glycohormones. These materials are known to contain several sugar chains in the molecule or microheterogeneity in single sugar chains. Therefore, it is necessary to fractionate these chains before structural analysis can be carried out.
Isolated oligosaccharides also are useful per se for preparing neoglycoproteins by attachment of individual oligosaccharides to various peptide or protein chains.
Hydrolytic digestion with enzymes, e.g. bacterial proteases such as Pronase.RTM., has long been a method of cleaving glycoproteins to obtain glycopeptides, but is generally unsatisfactory for various reasons including incomplete digestion and difficulties in fractionation of the resulting mixtures.
Chemical methods have also been employed to liberate free oligosaccharides from glycoproteins. A preferred chemical method involves hydrazinolysis which was originally described by Matsushima and Fujii, Bull. Chem. Soc. Jpn. 30, 48 (1959), and later developed by Bayard and Montreuil, "Methodologie de la Structure et du Metabolisme des Glycoconjugues," pp. 208-18, CNRS, Paris, 1974. According to the latter published procedure, the glycoprotein was heated with anhydrous hydrazine for 30 hours followed by treatment with nitrous acid to fragment the oligosaccharides. No attempt was made to recover intact oligosaccharides.
The hydrazinolysis method was further refined by Takasaki, Mizuochi and Kobata, Meth. Enzymol. 83, Academic Press, 1982, pp. 263-8. These investigators subjected the hydrazinolysis product to N-acetylation with acetic anhydride in saturated NaHCO.sub.3 solution followed by exchange of the Na.sup.+ with H.sup.+ ions by passage over a Dowex.RTM. 50 cation exchange resin. The N-acetylglucosamine residues were then subjected to paper chromatography using butanol:ethanol:water (4:1:1) and, after reduction with sodium borotritide (NaB.sup.3 H.sub.4) in NaOH, were subjected to another paper chromatographic step using ethyl acetate:pyridine:acetic acid:water (5:5:1:3). The released sugars were finally subjected to high resolution gel-permeation chromatography with Bio-Gel.RTM. P-4 to obtain an oligosaccharide profile as further described by Yamashita, Mizuochi and Kobata, Ibid., pp. 105-126. The Takasaki et al method, however, produces a mixture of degraded components containing reduced alditols. The reduction causes the production of radiolabeled reduced alditols, e.g. tritium-labeled N-acetylglucosaminitol or N-acetylmannosaminitol. According to Mellis and Baenziger, Anal. Biochem. 114, 276-280 (1981), the latter products are produced by alkali induced epimerization at the C-2 position of the amino-sugar at the reducing terminus. Such reduced alditols are not suitable for further reactions.
Although the Takasaki et al. method is useful for structural analysis of glycoproteins, the published procedure is not practical for a preparative scale isolation of unreduced oligosaccharides. It also results in substantial loss of sialic acid residues.